1. Field of the Invention
The present invention relates to a pknB kinase and a pstP phosphatase as well as their use for identifying antibacterial substances.
2. Description of the Background
Tuberculosis (TB) is a major public health problem with one-third of the world's population infected by its aetiologic agent, Mycobacterium tuberculosis, and over two million people dying from the disease each year (Dye et al., 1999, WHO Global Surveillance Monitoring Project J Am Med Assoc 282: 677-686). The Global Alliance for TB Drug Development has proposed that the current treatment could be improved considerably by developing more potent therapeutic agents, that reduce the duration of therapy, and by including drugs that act on latent bacilli (Global Alliance for TB Drug Development. (2001) Scientific blueprint for tuberculosis drug development. Tuberculosis 81: 1-52.). Faced with the urgency to develop new therapeutic strategies, it appears crucial to understand better the physiopathology of the causative agent and its complex relationship with the immune system of the host.
After inhalation, infectious bacilli are phagocytosed by alveolar macrophages in the lung and induce a local pro-inflammatory response, which leads to the recruitment of monocytes from the bloodstream into the site of infection (Dannenberg, A. M. (1999) Pathophysiology: basic aspects. In Tuberculosis and Nontuberculous Mycobacterial Infections. Schlossberg, D., (ed.). Philadelphia: W.B. Saunders Company, pp. 17-47; Russell, 2001, Nature Rev Mol Cell Biol 2: 569-577). By blocking fusion of phagosomes with lysosomes in these non-activated macrophages (Brown et al., 1969, Nature 221: 658-660; Sturgill-Koszycki et al., 1996, EMBO J 15: 6960-6968), M. tuberculosis escapes killing and multiplies. As the immune response progresses, macrophages and T cells accumulate to form a granuloma in which the pathogen is contained in a latent state (Parrish et al., 1998, TIBS 6: 107-112; Manabe and Bishai, 2000, Nature Med 6: 1327-1329). It can lie dormant for years only to rise again when the immune system wanes through old age, malnutrition or AIDS (acquired immuno-deficiency syndrome). The centre of the granuloma then liquefies and M. tuberculosis replicates profusely and is discharged into the bronchial tree producing an infectious cough (Dannenberg, 1999, Pathophysiology: basic aspects. In Tuberculosis and Nontuberculous Mycobacterial Infections. Schlossberg, D., (ed.). Philadelphia: W.B. Saunders Company, pp. 17-47). To understand the bacterial response to these changes in host environment, the study of regulatory proteins involved in mycobacterial signal transduction is therefore of the utmost importance.
Phosphorylation, a simple and efficient means of reversibly changing the biochemical properties of a protein, is a major mechanism for signal transduction and regulation of almost all biological functions. There are two main phosphorylative signal transduction systems. Prokaryotes predominantly use the two-component system, comprising in its simplest form a signal sensor with a histidine kinase domain and a response regulator, often a transcriptional factor (Wurgler-Murphy and Saito, 1997, TIBS 22: 172-176; Stock et al., 2000, Annu Rev Biochem 69: 183-215). This simple, unidirectional mechanism allows a quick response to abrupt environmental changes. The second system depends on the reversible phosphorylation of serine, threonine and tyrosine residues, and is widely used in eukaryotes (Hanks and Hunter, 1995, FASEB J 9: 576-596; Hunter, 1995, Cell 80: 225-236; Barford et al., 1998, Annu Rev Biophys Biomol Struct 27: 133-164; Hunter, 2000, Cell 100: 113-127). This mechanism involves the action of protein kinases and phosphoprotein phosphatases in cascades and networks (Hunter, 2000, Cell 100: 113-127), providing an efficient means for the rapid modulation of the transduced signal to serve highly regulated functions.
Since the identification of the first bacterial homologue a few years ago (Muñoz-Dorado et al., 1991, Cell 67: 995-1006), genomics has now demonstrated that serine, threonine and tyrosine protein kinases and phosphatases are also widespread in prokaryotes (Zhang, 1996, Mol Microbiol 20: 9-15; Kennelly, 2002, FEMS Microbiol Lett 206: 1-8). The two phosphorylation mechanisms (two-component systems and Ser/Thr/Tyr kinases and phosphatases) in prokaryotes may regulate distinct functions or act together in the same signalling pathway. The presence of Ser/Thr and Tyr kinases and phosphatases in prokaryotes appears to be associated with a complex, multistage developmental cycle and possible roles in regulating growth and development (heterocyst, fruiting-body or spore formation) have been proposed (Zhang, 1996, Mol Microbiol 20: 9-15; Shi et al., 1998, FEMS Microbiol Rev 22: 229-253). The dormant state of M. tuberculosis, although poorly understood, may be considered in some regards analogous to sporulation (Demaio et al., 1996, Proc Natl Acad Sci USA 93: 2790-2794) and thus involve these enzymes.
Mycobacterium tuberculosis employs both systems of protein phosphorylation. It has 15 sensor His kinases and 15 response regulators, forming at least 11 functional pairs, together with 11 putative Ser/Thr protein kinases (STPKs), one phospho-Ser/Thr phosphatase (ppp renamed here pstP) and two Tyr phosphatases (ptpA, ptpB) (Cole et al., 1998, Nature 393: 537-544). There appears to be no counterpart Tyr kinase for the two Tyr phosphatases, PtpA and PtpB, which can, moreover, be secreted (Koul et al., 2000, Microbiology 147: 2307-2314; Cowley et al., 2002, Res Microbiol 153: 233-241). Eight of the 11 STPKs are predicted to be transmembrane proteins, with a putative extracellular signal sensor domain and an intracellular kinase domain. Six STPKs (PknA, B, D, E, F, G) have already been expressed as recombinant proteins and shown to be functional kinases (Peirs et al., 1997, Eur J Biochem 244: 604-612; Av-Gay et al., 1999, Infect Immun 67: 5676-5682; Koul et al., 2001, J Bacteriol 182: 5425-5432; Chaba et al., 2002, Eur J Biochem 269: 1078-1085; data not shown for PknE).
At this time, no physiological role has been clearly demonstrated for any of the STPKs or phosphatases from M. tuberculosis, and knock-out mutants have not yet been reported.
In view of the above, there remains a need for developing new targets and therapies for mycobacterial infections.